Using short-read 16S rRNA sequencing of multiple variable regions to generate high-quality results to a species level

Amy S Graham ^1,2* Fadheela Patel ³ Francesca Little ⁴ Andre van der Kouwe ^5,6 Mamadou Kaba ³ Martha J Holmes ^1,2,7,8

• ¹ Imaging Sciences, Neuroscience Institute, University of Cape Town, Cape Town, South Africa
• ² Department of Human Biology, Division of Biomedical Engineering, University of Cape Town, Cape Town, South Africa
• ³ Department of Pathology, Division of Medical Microbiology, University of Cape Town, Cape Town, South Africa
• ⁴ Department of Statistical Sciences, University of Cape Town, Cape Town, South Africa, Cape Town, South Africa
• ⁵ Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, United States
• ⁶ Department of Radiology, Harvard Medical School, Boston, United States
• ⁷ Department of Biomedical Physiology and Kinesiology, Faculty of Science, Simon Fraser University, Burnaby, British Columbia, Canada
• ⁸ SFU Imagetech Lab, Simon Fraser University, Surrey, British Columbia, Canada

Introduction Short-read amplicon sequencing studies have typically focused on 1-2 variable regions of the 16S rRNA gene. Species-level resolution is limited in these studies, as each variable region enables the characterisation of a different subsection of the microbiome. Although long-read sequencing techniques can take advantage of all 9 variable regions by sequencing the entire 16S rRNA gene, short-read sequencing has remained a commonly used approach in 16S rRNA research. This work assessed the feasibility of accurate species-level resolution and reproducibility using a relatively new sequencing kit and bioinformatics pipeline developed for short-read sequencing of multiple variable regions of the 16S rRNA gene. In addition, we evaluated the potential impact of different sample collection methods on our outcomes. Methods Using xGenTM 16S Amplicon Panel v2 kits, sequencing of all 9 variable regions of the 16S rRNA gene was carried out on an Illumina MiSeq platform. Mock cells and mock DNA for 8 bacterial species were included as extraction and sequencing controls respectively. Within-run and between-run replicate samples, and pairs of stool and rectal swabs collected at 0-5 weeks from the same participants, were incorporated. Observed relative abundances of each species were compared to theoretical abundances provided by ZymoBIOMICS. Paired Wilcoxon rank sum tests and distance-based intraclass correlation coefficients were used to statistically compare alpha and beta diversity measures, respectively, for pairs of replicates and stool/rectal swab sample pairs. Results Using multiple variable regions of the 16S ribosomal Ribonucleic Acid (rRNA) gene, we found that we could accurately identify taxa to a species level and obtain highly reproducible results at a species level. Yet, the microbial profiles of stool and rectal swab sample pairs differed substantially despite being collected concurrently from the same infants. Conclusion This protocol provides an effective means for studying infant gut microbial samples at a species level. However, sample collection approaches need to be accounted for in any downstream analysis.